Recent studies by the Principal Investigator have demonstrated an unique control mechanism existing in mammalian cells, whereby the expression of the tubulin gene appears to be regulated by the level of unpolymerised tubulin in the cell. This is one of the first examples of a direct feedback mechanism, analogous to the bacterial systems, operating in higher eucaryotic cells. For this reason, much of the proposed work will be directed towards understanding this regulatory process. These studies will be extended to determine if such a mechanism also operates to modulate tubulin synthesis during different growth and/or differentiation states of various cells. It will be of particular interest to assess whether the synthesis of actin, another structural protein, responds in a similar fashion. Recently, it has been proposed that the cytoskeletal framework of cells serves a function to facilitate intracellular metabolism, in particular RNA metabolism and protein synthesis. The possibility that such a structure plays a major role in the regulation of tubulin and actin synthesis will also be addressed. The long term aim being: to test the hypothesis that the cytoskeletal framework acts as a mediator of signals, generated during the change in the configuration of a cell, to modulate the expression of tubulin and actin genes. The strategy designed to answer these questions is to manipulate cells (in particular 3T6 fibroblasts) in culture, in such a way as to alter their configuration in a controlled manner. These include: (i) drugs which depolymerise microtubules, (ii) suspension of cells in methocel, followed by reattachment to surfaces of differing adhesivity (including poly HEMA), (iii) synchronization of cells to study the cell cycle. The various levels of regulating tubulin and actin gene expression will then be analysed in these various cell configurations using specific cloned cDNA probes corresponding to the messenger RNAs coding for these cytoskeletal proteins. The approach will involve a systematic analysis of translation, mRNA function and stability in the cytoplasm, nuclear RNA processing and transcription of the specific sequences.